Proteins in solution are difficult to separate from one another, particularly with their biological activity preserved. The need for simple processes is great. Isolation of specific proteins is required for industrial processes and for biological and medical research, for example. Removal of harmful proteins from foods is also a frequent problem. Consequently, there has been enormous research toward the objective. Although many methods have been developed for specific separations, the procedures are frequently difficult and time-consuming, and in some cases there is no practical process. The search for better ways continues.
Membranes have been a popular approach in recent years. Their use does not involve extreme conditions, such as the high electrolyte concentrations frequently necessary in salting-out, that might denature the proteins. Because membranes are now available with controlled pore sizes of relatively narrow spreads, it would seem simple to exploit differences in protein sizes by choosing materials which pass one species and retain others. The situation is not so simple however. Proteins frequently adsorb on surfaces, and the adsorption can depend on pH, salt concentration and other characteristics of the medium, as well as the nature of the membrane material. Even without difficulties from specific adsorption on the surface of the pores, proteins or other substances in the medium may form a fouling layer, or dynamic membrane, at the interface and make the nominal pore size of the membrane irrelevant. Interaction of proteins in the solution phase can cause further complications. When pore dimensions are of the same order of magnitude as particle sizes, co interactions between particles and pores can significantly affect rejection, by ion ("Donnan") exclusion mechanisms. These influences may be complex, because of the variation of the sign and magnitude of protein charge with pH, as well as similar variations of membrane charge from pH or fouling.
In spite of these difficulties, separations with membranes have been realized. Cordle et al., in U.S. Pat. No. 4,897,465, claim to reduce the ratio of beta-lactoglobulin and alpha-lactalbumin to immuno-gamma globulin (gG) and bovine serum albumin (BSA) in cheese whey by passing the first two and retaining the others, in a single ultrafiltration and subsequent diafiltration (adding pH-adjusted water or physiological saline solution to retentate and removing it and other permeating impurities by further ultrafiltration). Their separation was carried out at pH 5. The membrane they used was a porous, stainless steel tube with a bed of metal oxide particles. They adjusted the pH to 7, and in a second ultrafiltration, passed gG and BSA with the permeate, thus purifying them from bacteria, fat, casein and other such large retained species. Although they did not demonstrate enrichment of BSA and gG relative to one another, they stated that they believed this would be feasible at pH 5.5. They also claimed the reverse procedure, the first ultrafiltration at pH 7 with the desired proteins in the permeate, freed of large unwanted species, and the second ultrafiltration at pH 5 , to concentrate gG and BSA and to remove unwanted small species in the permeate.
Central to their process was the concept that, with the filter they used, porous stainless steel with a layer of metal oxide particulates, proteins would be rejected at pH values below their isoelectric points and would permeate at pH values above. The isoelectric point (IEP) of gG is about 6.5 and of BSA about 4.8. The pH which they specify for expected (not demonstrated) separation is 5.5.
Ohno et al., in U.S. Pat. No. 4,347,138, were able to separate blood serum gamma globulin and albumin by ultrafiltration through a polysulfone membrane, stated to have a MW cutoff of 100,000. Large species had been removed prior to the ultrafiltration by centrifugation, and the volume was diluted to 1.5% total protein, salt added to 0.04M, and the pH adjusted to 4.1. Globulin was retained, but considerable albumin was passed when 80% of the volume had permeated. After several dilutions with buffer and subsequent ultrafiltration, most of the albumin was in permeate and most globulin in retentate.
Comparison of the recommended pH values in these two patents indicates that other factors must influence the results: Cordle et al. see no separation at pH 5, but believe that a more basic solution, pH 5.5, would be optimal, whereas Ohno et al. demonstrate separation at pH 4.1, on the acidic rather than the basic side of that used by Cordle, and specify a preferred range of 3.9-4.3. Differences in the membranes used and in bovine and human proteins are two of the many possible sources for differences in optimal separation conditions.
Roger et al. (in U.S. Pat. No. 4,485,040 and U.S. Pat. No. 4,711,953) fractionate alpha-lactalbumin in cheese whey by permeating it through a 50K MW cutoff membrane with pH adjusted to 6.6 to increase its passage, large species being retained; they then concentrated it and removed by low-molecular weight impurities by diafiltration through 2K MW cutoff membranes. Kolthe et al. (in U.S. Pat. No. 4,644,056) purify gG from milk or colostrum by diafiltering it through membranes of large pore size in a pH range of 4-5.5, and the permeate subjected to diafiltration with membranes of small pore size, low molecular weight species being removed. The pH range here at which gG permeates is the region used by Cordle et al. and Ohno et al. to retain it.
Although the specific differences in conditions responsible for the apparent contradictions in the teachings optimal for separations are not clear in all cases, the cited observations illustrate that protein passage and retention are complex phenomena, dependent on many factors.